人冠状病毒HKU1 S蛋白介导的细胞进入机制的研究

Human coronavirus (CoV) HKU1 is an important human pathogen causing the commom cold and other repsiratory diseases. Despite having made significant advances in our understanding of the epidemiology and clinical association of hCoV-HKU1, little is known regarding the biology or pathophysiology or entry mechanism of HKU1. Advances in the knowledge of HKU1 have been limited by the fact that it can only be grown in differentiated human airway epithelial (HAE) cells and very few reagents are available to study this virus. We recently have established primary HAE cell culture and differentiation system in our lab and successfully applied this technique to isolate and culture HKU1 virus. In addition, we screened and found several HKU1 S protein specific neutralizing mouse monoclonal antibody. In this study, we propose to map the epitope of neutralization antibodies, determine the receptor binding domain (RBD) of HKU1 S protein, and resolve the structure of RBD with or without neutralization antibody. By combination of HKU1 S lentiviral pseudotype and flow cytometry cell sorter techniques, we also plan to screen HAE cell cDNA expression library to determine the receptor for HKU1 virus. These discoveries will make significant contribution to our understanding the mechanism of HKU1 virus entry, pathogenesis, and coronavirus S protein evolution.

人冠状病毒HKU1是导致感冒等呼吸道疾病的一个重要病毒。虽然HKU1病毒的流行病学和疾病相关性的调查研究资料已经比较丰富和全面，但其感染机制等基础生物学知识仍非常匮乏。尤其是病毒的受体结合域及其细胞受体目前均属未知。阻碍HKU1病毒研究的主要因素是该病毒难以分离和培养。本课题组目前已初步建立了人呼吸道上皮细胞(HAE)体外培养分化体系，并且成功应用于HKU1病毒的分离和培养。本项目将应用这一体系，结合已拥有的HKU1 S蛋白特异的鼠单克隆抗体，通过抗体中和、蛋白质突变及竞争抑制感染等策略确定该病毒S蛋白的受体结合域的核心区和关键性氨基酸，进而解析其晶体结构。此外，我们将应用HKU1 S蛋白包裹的慢病毒体系和流式细胞分离技术对分化的HAE细胞的cDNA表达文库进行筛选以发现HKU1病毒的细胞受体。本研究将对阐明HKU1病毒的细胞感染机制,致病机理,以及冠状病毒S蛋白的进化提供重要信息。

人冠状病毒HKU1是一个重要的呼吸道病毒，其感染导致普通感冒，在老人、小孩以及免疫缺陷的人群中能导致肺炎，但有关HKU1病毒的基本生物学研究很少。经过四年的努力，本研究项目主要在以下几个方面取得了重要进展：第一、建立了成熟的人原代呼吸道上皮细胞的培养体系；第二、发现HKU1 S蛋白的受体结合区位于S蛋白的CTD区域；第三、解析了HKU1 S蛋白CTD的结构，并进一步找到了跟中和抗体结合5个关键氨基酸以及两个跟未知受体结合的重要氨基酸。有关HKU1病毒的受体的寻找，还在继续。这些成果填补了相关空白，极大增强我们对HKU1病毒的感染和免疫机制的了解，为进一步研究冠状病毒S蛋白的演化提供了有力帮助。